loading for western blot - (Apr/17/2007 )

Hi, looking people's advice for western blots, it seems that the problem is usually overloading. I'm wondering does overloading mean there is too much volume of sample or the protein concentration is too high? In terms of concentration, what is the general protein concentration that should be used for a western blot?

Thanks!

-The_Erotic_Terrorist-

I use 1 mg/mL and load 20 uL per lane, so it´s 20 ug of total protein per lane, although I have used 77 ug and had good bands but I think this is not common, so much protein causes band migration, unespecific bands, ...I think that around 20 ug is a good amount but let´s see what the others say.

-Pumuki-

Thanks

-The_Erotic_Terrorist-

The amount of protein depends on its purity.

If you have a highly purify protein, then load less amount (<10ug per lane).You probably may not want a big band on the western blot.

However, if not, then load more (>10ug, or even 50ug per lane). Or else, you may not see a band.

Hope this may help.

-Minnie Mouse-

Yeah it's not going to be pure. It;s going to be a western on conditioned media.

-The_Erotic_Terrorist-

QUOTE (The_Erotic_Terrorist @ Apr 27 2007, 01:10 PM)

Yeah it's not going to be pure. It;s going to be a western on conditioned media.

Still it would depends on the actual amount of the interested proteins in your sample. Also whether you are looking for a special protein or for many proteins which share a common epitope... would affect the actual result. So to have a good-looking WB, especially when you first work with a particular protein, you usually need to repeat several time looking for the right conditions. I would say that for media and your interested protein is not too low, maybe try 20ug per lane first. If not enough, then depends on the results, you may have to increase the sample volume or even TCA precipitate to increase the concentration. For medium, I normally try to have as concentrated as I can, especially with non-secretory cell lines

-Almasy-

QUOTE (Almasy @ Apr 27 2007, 09:04 AM)

QUOTE (The_Erotic_Terrorist @ Apr 27 2007, 01:10 PM)

Yeah it's not going to be pure. It;s going to be a western on conditioned media.

Still it would depends on the actual amount of the interested proteins in your sample. Also whether you are looking for a special protein or for many proteins which share a common epitope... would affect the actual result. So to have a good-looking WB, especially when you first work with a particular protein, you usually need to repeat several time looking for the right conditions. I would say that for media and your interested protein is not too low, maybe try 20ug per lane first. If not enough, then depends on the results, you may have to increase the sample volume or even TCA precipitate to increase the concentration. For medium, I normally try to have as concentrated as I can, especially with non-secretory cell lines

Hi. I agree with Almasy. In the westerns I'm doing I get a concentration of 6ug/ul and I load 10ul (i.e. 60ug of total protein) because the protein I am looking for is propably quite low. The blots i'm getting are background free and with only one unspecific zone. Of course after dying (after transfer) with ponceau I can see a lot of proteins on the membrane. I guess it depends n the protein you are looking for. good luck!

-charis-

I'm looking for a specific protein that I've transfected my 293H cells to produce. I've done a PCR and they've tested positive for mRNA expression. I've also done a bradford assay on the CM to get an idea how much stuff I'm working with and it seems that on average my samples have aproximately 480 microgram/ml of TOTAL protein. So buried somewhere in that sea of proteins are my proteins of interest. Thanks for the advice guys.